Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box protein^{S-Phase Kinase-Associated Protein 2B}-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses.Roots have developed adaptive strategies to reprogram their gene expression and metabolic activity in response to heterogeneous soil environments (Osmont et al., 2007). By this way, local environmental stimuli can be integrated into the developmental program of roots (Forde, 2014; Giehl and von Wirén, 2014). In resource-depleted environments, an important heterogeneously distributed soil factor is nutrient availability, which then directs lateral root growth preferentially into nutrient-rich patches (Zhang and Forde, 1998; Lima et al., 2010; Giehl et al., 2012). Such directed lateral root development depends on regulatory networks that integrate both local and systemic signals to coordinate them with the overall plant nutritional status (Ruffel et al., 2011; Guan et al., 2014). As shown by the impact of the N status-dependent regulatory module CLAVATA3/EMBRYO-SURROUNDING REGION-related peptides-CLAVATA1 leucine-rich repeat receptor-like kinase, economizing the costs for root development is pivotal for a resource-efficient strategy in nutrient acquisition (Araya et al., 2014). In recent years, strategies on yield and efficiency improvement have been developed that are primarily based on the manipulation of root system architecture (Gregory et al., 2013; Lynch, 2014; Meister et al., 2014). A common imperative of these strategies is to develop crops that use water and nutrients more efficiently, allowing the reduction of fertilizer input and potentially hazardous environmental contamination.Maize (Zea mays) plays an eminent role in global food, feed, and fuel production, which is also a consequence of its unique root system (Rogers and Benfey, 2015). The genetic analysis of maize root architecture revealed a complex molecular network coordinating root development during the whole lifecycle (for review, see Hochholdinger et al., 2004a, 2004b). Identification of root type-specific lateral root mutants in maize emphasized the existence of regulatory mechanisms involved in the branching of embryonic roots, which are distinct from those in postembryonic roots (Hochholdinger and Feix, 1998; Woll et al., 2005). Under heterogeneous nutrient supplies, nitrate-rich patches increased only the length of lateral roots in primary and seminal roots, whereas they increased both length and density of lateral roots from shoot-borne roots of adult maize plants (Yu et al., 2014a). Remarkably, modulation of the extensive postembryonic shoot-borne root stock has a great potential to improve grain yield and nutrient use efficiency (Hochholdinger and Tuberosa, 2009).Lateral root branching is critical to secure anchorage and ensure adequate uptake of water and nutrients. In maize, these roots originate from concentric single-file layers of pericycle and endodermis cells (Fahn, 1990; Jansen et al., 2012). Lateral root initiation is the result of auxin-dependent cell cycle progression (Beeckman et al., 2001; Jansen et al., 2013a). Most of the molecular changes during the cell cycle like, for instance, the induction of positive regulators, such as cyclins (CYCs) and cyclin-dependent kinases (CDKs), and the repression of Kip-related proteins (KRPs), thus account for a reactivation of the cell cycle (Beeckman et al., 2001; Himanen et al., 2002, 2004). In eukaryotes, ubiquitin-mediated degradation of cell cycle proteins plays a critical role in the regulation of cell division (Hershko, 2005; Jakoby et al., 2006). Conjugation of ubiquitin to a substrate requires the sequential action of three enzymes: ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase (E3). The E3 enzymes are responsible for the specificity of the pathway, and several classes of E3 enzymes have been implicated in cell cycle regulation, including the S-Phase Kinase-Associated Protein1-cullin-F-box protein (SCF) and Really Interesting New Gene (RING) finger-domain ubiquitin ligases (Del Pozo and Manzano, 2014). The F-box protein S-Phase Kinase-Associated Protein 2B (SKP2B) encodes an F-box ubiquitin ligase, which plays an important role in the cell cycle by regulating the stability of KRP1 and pericycle founder cell division during lateral root initiation (Ren et al., 2008; Manzano et al., 2012).It has been shown that auxin is involved in long-distance signaling to adjust root growth in response to local nutrient availability (Giehl et al., 2012), and it is likely to serve in long-distance signaling for local nutrient responses as well (for review, see Rubio et al., 2009; Krouk et al., 2011; Saini et al., 2013; Forde, 2014). Polar auxin transport is instrumental for the generation of local auxin maxima, which guide these cells to switch their developmental program (Vanneste and Friml, 2009; Lavenus et al., 2013). In Arabidopsis (Arabidopsis thaliana), the PIN-FORMED (PIN) family of auxin efflux carrier proteins controls the directionality of auxin flows to maximum formation at the tip or pericycle cells (Benková et al., 2003; Laskowski et al., 2008; Marhavý et al., 2013). Auxin responses in protoxylem or protophloem cells of the basal meristem coincide with the site of lateral root initiation (De Smet et al., 2007; Jansen et al., 2012). In these defined pericycle cells, the phloem pole pericycle founder cells are primed before auxin accumulation occurs (De Smet et al., 2007; Jansen et al., 2012, 2013a). In contrast to dicots, the larger PIN family in monocots has a more divergent phylogenetic structure (Paponov et al., 2005). It is likely that monocot-specific PIN genes regulate monocot-specific morphogenetic processes, such as the development of a complex root system (Wang et al., 2009; Forestan et al., 2012).The molecular control of lateral root initiation of the root system to heterogeneous nitrate availabilities is not yet understood in maize. In this study, the plasticity of lateral root induction in adult shoot-borne roots of maize in response to local high concentration of nitrate was surveyed in an experimental setup that simulated patchy nitrate distribution. RNA-sequencing (RNA-Seq) experiments and cell type-specific gene expression analyses showed that local nitrate triggers progressive cell cycle control during pericycle cell division. In addition, tissue-specific determination of indole-3-acetic acid (IAA) and its metabolites combined with auxin maxima determination by DR5 supported a role of basipetal auxin transport during lateral root initiation in shoot-borne roots. Thereby, this study provides unique insights in how auxin orchestrates cell cycle control under local nitrate stimulation in the shoot-borne root system of maize.